Lentiviral Knockdown and Overexpression of Skp2 and MLKL

Lentivirus plasmids containing pLKO.1-shSkp2 were purchased from Thermo Scientific. The pLKO.1-shGFP (plasmid #30323), the lentiviral packaging plasmid psPAX2 (plasmid #12260) and the envelope plasmid pMD2.G (plasmid #12259) were available on Addgene (Cambridge, MA). The expression constructs pCMV6-Entry-Skp2 (#RC214001) and pCMV6-Entry-MLKL (#RC213152) were purchased from OriGene (Rockville, MD). The MLKL expression construct pEGFP-MLKL was purchased from Youbio (Changsha, CN). The generation of gene stable knocking down NCI-H1299, NCI-H23, NCI-H125 or A549 cell lines was performed as described previously^{77 (link)}. For transient expression of MLKL or Skp2 by plasmid transfection, cells in 6-well plates were transiently transfected with 2.0 μg construct with Lipofectamine 2000 (#11668-019, Invitrogen, Carlsbad, CA) for 48 h following the manufacturer’s instructions. RNA interference was performed as described previously^{77 (link)}. Cells were grown in 6-well plates and transfected with 100 pmol Control siRNA (#6568), Skp2 siRNA I (#7753), Skp2 siRNA II (#7756) or siMLKL (sc-93430) using HiPerFect transfection reagent (#301705, Qiagen) for 72 h according to the manufacturer’s instructions and the knockdown of Skp2 was confirmed by Western blot analysis.